HIV-1 encodes a number of genes that are crucial for replication in primate cells. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest are the HIV accessory proteins Vif, Vpr, Vpu, Vpx, and Nef, which are unique to primate lentiviruses. There is increasing evidence that these proteins operate in conjunction with specific host factors. In fact, most if not all, of the accessory proteins lack catalytic activities but instead seem to function as adaptors to link viral or cellular factors to pre-existing cellular pathways. In FY 2011, we continued studies to improve our understanding of the functional interactions between HIV-1 Vif and the host restriction factor APOBEC3G (A3G) and related enzymes, in particular A3F and A3H. We identified dominant negative mutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3F and A3H. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant negative phenotype. Expression of dominant negative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominant negative Vif interfered with wild-type Vifs ability to exclude A3F and A3H from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies. During FY2011 we also continued our analysis of Vpu and its functional interaction with the virus-release antagonist BST-2/tetherin. We continued a study initiated in FY2010 studying the effects of Vpu on BST-2 stability. The idea to this project is in part based on the observation that Vpu very efficiently induces the degradation of CD4 through an ER-associated degradation (ERAD) pathway and in part on reports by other labs describing an increased turnover of BST-2 in the presence of Vpu. We performed metabolic labeling and pulse/chase analysis of endogenous BST-2 in various cell types and found that Vpu only modestly increased the turnover of a specific subset of BST-2. Surprisingly, the effect of Vpu on transiently expressed BST-2 differs from that described for endogenous BST-2. In the case of transiently expressed BST-2, we observed ERAD-dependent degradation in the presence of Vpu. However, in this case Vpu targeted a different subset of BST-2 molecules than in the experiments involving endogenous BST-2. These results suggest that the molecular mechanisms leading to the increased turnover of endogenously and exogenously expressed BST-2 are distinct. Part of this project is now published (Andrew 2011). We also continued a project to investigate the effect of deletions or insertions in the BST-2 ectodomain on interference with virus release. This project was in part stimulated by reports showing that synthetic constructs can have BST-2-like function as long as certain structural features are preserved. A large portion of the BST-2 ectodomain is predicted to assume a coil-coil structure. We found the insertions or deletions in this domain can either be tolerated with little impact on BST-2 function or can completely abrogate BST-2s ability to antagonize virus release. Our initial data suggest that the effect of a deletion or insertion is more dependent on the size of the deletion/insertion rather than the location in the molecule. We hypothesize that insertions or deletions in the BST-2 ectodomain impose structural constraints that may or may not affect protein function. To test our hypothesis we are currently performing molecular modelling studies to investigate the effects on our mutations on BST-2 structure. Finally, we initiated a project to investigate the presumed GPI anchor modification of BST-2. A current model suggests that BST-2 tethers mature virions to the cell surface by means of its N-terminal TM domain and C-terminal GPI anchor. Indeed, immune electron microscopy confirmed that BST-2 could be found on virions tethered to the cell surface. However, the evidence for BST-2 containing a GPI anchor in addition to a transmembrane domain mainly consists of experimental data from the rat system. While all GPI anchored proteins are initially synthesized with a TM domain, the vast majority of mature GPI anchored proteins lack a TM domain making BST-2 one of only few proteins carrying a TM domain in addition to a GPI anchor. In fact, only four other naturally occurring proteins are known to be anchored in the membrane by both a TM domain and a GPI anchor. Experimental verification of GPI anchor modification of proteins containing an additional TM domain is technically challenging. We used a variety of biochemical assays including PI-PLC treatment, aerolysin treatment, and gradual truncation of the putative GPI anchor signal. We were unable to verify GPI anchor modification of human BST-2. Instead, we found strong evidence that the C-terminal putative GPI anchor signal in human BST-2 represents, in fact, a second TM domain. This conclusion is supported by the following observations: (i) the C-terminal putative GPI anchor domain can be transferred to a heterologous protein and function as a TM domain;(ii) C-terminally epitope-tagged BST-2 is functional. Importantly, the C-terminal tag was not subject to proteolytic removal by the GPI modification machinery and localized to the cytoplasmic side of the plasma membrane; (iii) deletion of 2 C-terminal residues from untagged BST-2 does not affect BST-2 function but can be visualized as a shift in protein mobility arguing against proteolytic removal of the GPI signal peptide;(iv) replacing the GPI anchor domain in human BST-2 with the TM domain of heterologous proteins can yield BST-2 capable of inhibiting virus release.